Hela cell nucleosomes have been prepared as a highly homogeneous population containing 140 plus or minus 5 base pairs of DNA. During the digestion of nuclear DNA to produce nucleosomes, a 60-70 base pair bridging DNA region between nucleosomes is destroyed. Histone H1 appears to bind to the bridging DNA. The nucleosomes contain 1.2 gm histone/gm DNA. Histone H2B reacts with specific anti-H2B antibody while complexed in the nucleosome. Immunosedimentation demonstrates that all nucleosomes contain identical complements of histones, two each of H2A, H2B, H3 and H4. Physicochemical characterization of DNA structure within the nucleosome has been carried out. The role of histone-histone interactions in stabilization of DNA conformation in nucleosomes has been evaluated by solvent perturbation, thermal denaturation and protein cross-linking experiments. The DNA of intact nucleosomes can be labeled at the 5 feet termini with ATP and polynucleotide kinase. Using labeled particles, we show that there is a potential single strand nick site for DNase I every ten nucleotides along the DNA within nucleosomes - these sites vary widely in their actual susceptibility to the nuclease. Chemical modification studies of histones in chromatin are consistent with models for nucleosome structure which envision hydrophobic interactions between the C-terminal portions of the histones and electrostatic DNA-amino terminal binding.